British Journal of Pharmacology and Toxicology 4(2): 25-32, 2013
ISSN: 2044-2459; e-ISSN: 2044-2467
© Maxwell Scientific Organization, 2013
Submitted: May 15, 2012 Accepted: December 28, 2012 Published: April 25, 2013
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Abstract: Chemotherapy of African trypanosomiasis in both the human and animal forms has been confronted with multidimensional problems that include paucity of drugs, resistance, high cost, prolonged treatment protocol and adverse side effects. The main objective of this study was to explore alternative sources of antitrypanosomal agents from the leaves, stem and root barks of Eucalyptus camaldulensis.
The plant parts were sequentially extracted with hexane, ethyl acetate, methanol and water; and the extracts screened for antitrypanosomal activity. Mice infected with Trypanosoma brucei brucei were administered intraperitoneally doses ranging from 200-600 mg/kg body weight/day of the extracts for 21 consecutive days. One control group was treated with 3.5mg/kg bodyweight of berenil while the other control group was left untreated. The methanol extract of E. camaldulensis (leaf) produced complete cure for the animals in the different dose groups and survived as long as those treated with the standard drug, berenil, although the clearance time was faster for the standard drug. Sub inoculation of healthy mice with the blood and Cerebrospinal Fluid (CSF) of the cured mice did not result in infection, thus indicating a complete and permanent cure. Acute toxicity studies of the methanol extract of E. camaldulensis (leaf) confirmed the safety of the extract because no mortality was recorded even at 5000 mg/kg bodyweight. However, the extract had no prophylactic activity. Bioassay-guided fractionation of the crude methanol extract of E. camaldulensis leaf gave 10 fractions, with only fractions 8 and 9 exhibiting minimal antitrypanosomal activities that were not comparable to those of the crude extract and the standard drug (p≤0.05). Phytochemical screening revealed the presence of terpenes, steroids, saponins, tannins, alkaloids and fatty acids in both the crude extract and fraction 9; while fraction
8 contained only terpenes, steroids and fatty acids. Data from GC-MS analysis of the two fractions indicated likely components to be methyl esters, amides, long chain alkenes and alcohols. The result of this study shows that the methanol extract of E. camaldulensis (leaf) has immense potential for the development of drugs against African trypanosomiasis.
Keywords: Antitrypanosomal, cerebrospinal fluid, Eucalyptus camaldulensis , methanol extract, Trypanosomiasis
INTRODUCTION
Human African trypanosomiasis, also known as sleeping sickness, is a vector-borne parasitic disease.
The parasites concerned are protozoa belonging to the
Trypanosoma genus. They are transmitted to humans by tsetse fly ( Glossina genus) bites which have acquired their infection from human beings or from animals harboring the human pathogenic parasites.
The disease affects mostly poor populations living in remote rural areas of Africa. Untreated, it is usually fatal. Travellers also risk becoming infected if they venture through regions where the insect is common.
Generally, the disease is not found in urban areas, although some cases have been reported in suburban areas of big cities in some disease endemic countries
(Simarro et al ., 2011).
Sleeping sickness occurs only in 36 sub-Saharan
Africa countries where there are tsetse flies that can transmit the disease. In 1995, WHO Expert Committee estimated that 60 million people were at risk with an estimated 300 000 new cases per year in Africa, with fewer than 30,000 cases diagnosed and treated (WHO,
2012).
After continued control efforts, the number of cases reported in 2009 has dropped below 10 000 for first time in 50 years. This trend has been maintained in
2010 with 7139 new cases reported (WHO, 2012).
Corresponding Author: Y.A. Kabiru, Department of Biochemistry, Trypanosomiasis and Malaria Research Unit, Federal
University of Technology, Minna, Nigeria
25

Br. J. Pharmacol. Toxicol., 4(2): 25-32, 2013
Diagnosis and treatment of the disease is complex and requires specifically skilled staff. cultivated the world over for its oil, gum, pulp, timber, medicine and aesthetic value. Among the various wood and non-wood products, essential oil found in its Most of the drugs in use were developed decades ago and show variable efficacy, serious side effects, can require long-term treatment and many have poor activity in immuno-suppressed patients. These picture/prospects clearly indicate the necessity to search foliage is the most important one and finds extensive use in food, perfumery and pharmaceutical industry. In addition, the oil possesses a wide spectrum of biological activity including anti-microbial, fungicidal, for new chemotherapeutic agents (Legros et al ., 2002).
Relapse rate with melarsoprol in Northern Uganda,
Southern Sudan and Northern Angola was as much as insecticidal/insect repellent, herbicidal, acaricidal and nematicidal (Batish et al ., 2008). Eucalyptus oil is readily steam distilled from the leaves and can be used for cleaning, deodorising and in very small quantities in 30% (Ogbadoyi et al ., 2007) and most of the available drugs are highly toxic with about 5% of those treated with melarsoprol dying as a result of high toxicity of the drug (Ogbadoyi et al ., 2007). In addition to these food supplements; especially sweets, cough drops and decongestants. Eucalyptus oil has insect repellent properties and is an active ingredient in some commercial mosquito repellents (Doran and Brophy, drawbacks is the problem of paucity of the drugs in rural communities where the burden of the disease is more manifest and when the drugs are available, the cost is prohibitive. Despite efforts made by the World
1990).
Based on the fore-going and the fact that
Eucalyptus camaldulensis (Myrtaceae) leaves are used singly and in combination with some other plant parts Health Organization (WHO) to help find new treatments, there have been few new drugs in recent times. Anti-trypanosomiasis vaccination still remains the best theoretical option in the fight against a disease to treat malaria and typhoid fevers in some Northern parts of Nigeria, this study was designed to evaluate the anti-trypanosomal potential of the plant. that is continuously hovering between its wildlife reservoir and its reservoir in man and livestock. While antigenic variation of the parasite surface coat has been considered the major obstacle in the development of a Materials:
MATERIALS AND METHODS
Plant materials: The leaves of Eucalyptus functional vaccine, recent research into the biology of B cells has indicated that the problems might go further than that (Magez et al ., 2010). As a consequence of these apparent setbacks and the non accessibility and
Camaldulensis were collected from the Education
Resource Center/Education Trust Fund ground in
Minna, Niger State, Nigeria, between the months of
June and July, 2009. The specimen was identified and affordability of the few available trypanocidal drugs, majority of the world’s population especially in Africa depend on traditional medical remedies for this infection and it is estimated that some 20,000 species of allotted a voucher number (NIPRD/H/6263) at the
National Institute for Pharmaceutical Research and
Development, Abuja, Nigeria.
All aspects of this study between 2009 and 2011 were carried out in the higher plants are used medicinally throughout the world
(Phillipson and Wright, 1991).
Many Traditional medical remedies have been used extensively and knowledge about them has been
Biochemistry laboratory of Federal University of
Technology, Minna, Nigeria, except for the fractionation that was done in the plant chemistry laboratory of National Institute for Pharmaceutical accrued by several generations of practitioners from experience, trial and error. Although formal toxicology studies are limited, most of the extensively used local remedies are unlikely to be severe toxins and are
Research and Development, Abuja, Nigeria.
Experimental animals: Albino mice, used for screening, were purchased from the Biochemistry and worthy of further evaluation for novel antiparasitic compounds (Swerdlow, 2000). Ethno medicine and ethno botany have long been of interest to medical researchers, physicians, the pharmaceutical industry,
Chemotherapy division of the National Institute for
Trypanosomiasis and Onchocerciasis Research, Vom,
Plateau State, Nigeria. The animals were bred in the
Department of Biochemistry laboratory, Federal anthropologists and botanists (Phillipson and O, Neill,
1986; Phillipson and Wright, 1991). And because greater importance is now being attached to the use of locally available medicines as a means of reducing
University of Technology, Minna, for subsequent use.
All experiments involving the animals were conducted in compliance with the internationally accepted principles for laboratory animal use and care as reliance on expensive imported drugs (Bodeker and
Willcox, 2000), the need to investigate unexplored natural products for their medicinal properties with contained in the Canadian Council on Animal Care guidelines on animal use protocol review (1997) and as described by Adamu et al . (2010). some urgency cannot be over-emphasized.
Eucalyptus camaldulensis (Myrtaceae), an
Australian native, represented by around 700 species is a genus of tall, evergreen and magnificent trees
Trypanosoma brucei brucei
Trypanosoma brucei brucei
: A stabilate of
(Lafia strain) was obtained
26

Br. J. Pharmacol. Toxicol., 4(2): 25-32, 2013 from the National Institute for Trypanosomiasis and
Onchocerciasis Research, Vom, Plateau State, Nigeria and subsequently maintained in the laboratory of the
Biochemistry Department, Federal University of
Technology, Minna, Niger State, Nigeria, by serial mg/kg body weight per day of the extracts respectively for 21 consecutive days commencing 24 h after infection. Group D was infected but not treated to serve as negative control, while Group E was infected and administered a single dose of 3.5 mg/kg bodyweight of berenil to serve as positive control. passage in mice. The major part of this study was conducted in the Department of Biochemistry
Laboratory, Federal University of Technology, Minna, while fractionation was done in the National Institute for Pharmaceutical Research and Development, Abuja,
Nigeria between 2010 and 2011.
Methods:
Preparation of plant samples: About 500 g of the leaves of the leaves of E. camaldulensis were removed fresh, washed with running tap water and dried at room temperature.
The dried leaves were blended into powdered form using mortar and pestle and subsequently stored in clean polythene bags until required for use.
Preparation of crude extracts: The extract was prepared and screening carried out using the method described by Adamu et al . (2010). In this method, fifty grams (50 g) of the dried powdered leaves was extracted under reflux with 400 ml of methanol for 2 h.
The extract was filtered hot using cheese cloth and solvent was removed using rotary evaporator followed by drying on steam bath. The extract, now in a relatively dry form, was transferred into a sterile sample bottle for storage in the refrigerator until required for use.
Infection of animals: The animals were inoculated using the method described by Mann et al . (2011).
Blood from a highly infected mouse was obtained by cardiac puncture and collected with EDTA-coated
Determination of minimum curative dose of the methanol extract of E. camaldulensis leaf: The methanol extract of E. camaldulensis (leaf), based on the anti-trypanosomal potential exhibited in the preliminary screening, was subjected to determination of the minimum dose that will produce a complete cure for T. b. brucei four weeks.
extract of
- infected mice. Six groups of mice, each group consisting of three mice, were set up.
Groups A, B, C and D were infected with and treated with 50,100, 150 and 200 mg/kg bodyweight per day of the extract for 21 days. Group E was infected and left untreated, while Group F was infected and administered a single dose of 3.5 mg/kg body weight of berenil. Parasitemia was monitored for
Blood and Cerebrospinal Fluid (CSF) infectivity tests: Both tests were carried out according to the method described by Ugwu et al completely cleared of parasites.
. (2011). Blood from animals that survived after treatment with the methanol
E. camaldulensis healthy mice in order to rule out the possibility of infection and to ascertain that cured mice were
Blood infectivity test:
T. b. brucei
leaf was used to inoculate dose groups treated and cured with crude methanol extract of E.
One mouse from each of the camaldulensis (leaf) was sacrificed 12 syringe. The blood was appropriately diluted with physiological saline to serve as inoculum. Healthy mice of weight range 25-35 g were infected intraperitoneally with 0.1 ml of the inoculum containing about 10
3 trypanosomes. weeks post-treatment and 0.02 ml of diluted blood obtained from the punctured heart of each mouse was sub-inoculated into clean parasite-free mice (two in each group) and parasitemia was monitored over a 2month period.
Cerebrospinal Fluid (CSF) infectivity test: One
Preparation of stock solution of extract: The stock solution was prepared just before use by dissolving the methanol extract in 10% Dimethylsulfoxide (DMSO).
Administration of crude extract and monitoring the course of parasitemia: The course of parasitemia was monitored using the method described by Adamu et al .
(2010). The presence of parasites in experimental animals was monitored three times weekly by making a blood smear on a glass slide and observing under a mouse from each of the dose groups treated and cured with crude methanol extract of E.
camaldulensis (leaf) had the hair on the back carefully shaved and was positioned such that the head touched the limbs. This made the vertebrae conspicuous for easy insertion of needle. The lumbar was then punctured by the insertion of a clean micro needle and syringe, into which a Clean,
Clear and Transparent Fluid (CSF) flowed. This was done under a mild anaesthesia and gentle handling. For each of the mouse from the treated groups, two clean, microscope set at 40X magnification. The number of parasites in 1 ml of blood was then estimated using the method of Herbert and Lumsden (1976).
The methanol parasite-free mice were sub-inoculated with 0.02ml of the CSF and parasitemia was monitored over a 2- month period. extract of E. camaldulensi (leaf), was screened for antitrypanosomal activity using five groups; with each consisting of three infected mice. Groups A, B and C were administered intraperitoneally 200, 400 and 600
27
Screening for prophylactic activity of extract: The methanol extract of E. camaldulensis leaf was screened for prophylactic activity using a modified method of

3.5
3
2.5
2
1.5
1
0.5
Br. J. Pharmacol. Toxicol., 4(2): 25-32, 2013
A-200mg/kgBW
B-400mg/kgBW
C-600mg/kgBW
D-Inf.NT
E-Inf.Treated(3.5mg/kgBW berenil)
0
1 2 3 6 10 15 17 20 30 40 60
-0.5
-1 Days post infection
Fig. 1: Course of parasitemia in infected mice treated with crude methanol extract of E. camaldulensis (leaf), treatment with 200,
400 and 600 mg/kg Body Weight (BW) of the extract cleared parasites from infected mice 10 days into treatment as did the standard drug (berenil)
Mgbemena et al . (2010). Two groups of mice, each group consisting of five mice, were set up. Animals in
Group A were administered 200 mg/kg bodyweight of methanol extract of E. camaldulensis while those in
Group B were administered 0.1ml of physiological saline for five consecutive days. On the sixth day, animals in the two groups were inoculated with 0.1ml of diluted trypanosome - infected blood (approximately
10
3 trypanosomes/ml). Parasitemia was thereafter analysis using standard analytical methods described by
Ugwu et al . (2011).
Gas Chromatography-Mass Spectroscopy (GC-MS) analysis: Fractions 8 and 9 were subjected to Gas
Chromatography-Mass Spectroscopy (GC-MS) in order to identify likely structures present in the two fractions.
The GC-MS spectrums were obtained using GCMS-
QP2010 PLUS (SHIMADZU, JAPAN).
monitored twice weekly for two weeks.
Acute toxicity studies of methanol extract of
E. camaldulensis (leaf): The acute toxicity of the crude
Statistical analysis of experimental data: Data obtained in this study were subjected to a one-way analysis of variance (ANOVA) to derive mean values methanol extract of E. camaldulensis leaf was evaluated using the method described by Ugwu et al . (2011). Six groups, consisting of three mice each, were administered single doses of 1000, 2000, 3000, 4000 of parasitemia which were compared with least significant difference. Mean values among the treated groups were deemed to be significantly different if the level of probability was ≤0.05.
RESULTS and 5000 mg/kg body weight of the extract respectively. The control group was administered 0.4 mL of physiological saline. Signs of acute toxicity such as death, changes in physical appearance and behavioural change were monitored over a two-week period.
Bioassay-guided fractionation of methanol extract of
Antitrypanosomal activity of crude methanol extract of E.camaldulensis (leaf): All animals in the three dose groups (200, 400 and 600mg/kg bodyweight/day) had parasites cleared from circulation about 2 weeks into
E. camaldulensis leaf: Ten fractions obtained from column chromatography were screened for antitrypanosomal activity. For each of the fractions, three groups, consisting of three mice each, were set up.
Doses of 50, 100 and 150 mg/kg body weight were administered on T. b.
brucei -infected mice in groups 1, the treatment period and this was maintained beyond 40 days (Fig. 1). The anti-trypanosomal activity of the extract was not significantly different from that of the standard drug, berenil (p
≤0.05). Animals in the untreated group started dying 10 days post-infection with parasitemia rising to about 0.3 x 10
6
/ml. of blood.
2 and 3 respectively and parasitemia was monitored for two weeks.
Minimum curative dose of methanol extract of
E. Camaldulensis (leaf): The best anti- trypanosomal activity was observed in the group treated with
Phytochemical analysis: The crude methanol extract
200mg/kg bodyweight indicating that it was the of E. camaldulensis (leaf) and two moderately active fractions (8 and 9) were subjected to phytochemical minimum dose that can clear blood stream trypanosomes in the test animal (Fig. 2).
28

1.5
1
0.5
2.5
2
Br. J. Pharmacol. Toxicol., 4(2): 25-32, 2013
A-50mg/kgBW
B-100mg/kgBW
C-150mg/kgBW
D-200mg/kgBW
E-Inf.Not Trt
F-Inf.Trt (3.5mg/kgBW)
0
1 2 3 6 10 15 17 20 30 40
-0.5
Days post infection
Fig. 2: Determination of minimum curative dose for crude methanol extract of E. camaldulensis (Leaf)
0.6
0.5
A-200mg/kgBW(Crude MeoH ext. Of
E.C Leaf)
B-150mg/kgBW(Fraction 8)
C-150mg/kgBW(Fraction 9)
0.4
D-Inf.NT
0.3
E-Inf.Treated(3.5mg/kgBW berenil)
0.2
0.1
0
-0.1
1 3 5 7 9 13 15 30
-0.2
Days post infection
Fig. 3: Course of parasitemia in infected mice treated with crude methanol extract of E. camaldulensis (leaf), fractions 8, 9 and standard drug, berenil; the two fractions succeeded in reducing the parasitemia but did not completely clear parasites from circulation
Blood and cerebrospinal fluid infectivity tests: All animals sub-inoculated with blood and cerebrospinal no record of death. Initial signs of weakness and difficulty in feeding were reversed on the same day . fluid from cured mice failed to come down with
T. b. brucei infection thus ascertaining the complete clearance of parasites by the crude methanol extract of
Prophylactic activity of crude methanol extract of
E.camaldulensis: after two months of monitoring,
E.camaldulensis (leaf).
The extract had no prophylactic
Antitrypanosomal activity of Fractions: Only fractions 8 and 9 at a dose of 150 mg/kg bodyweight/day extended the lifespan of animals by 6 days beyond the untreated infected control and within the treatment period, parasitemia was reduced considerably, while the animals treated with the crude methanol extract of E. camaldulensis and the standard activity because infected mice exhibited high levels of parasitemia and died within one week.
Acute toxicity test: All animals administered 1000-
5000 mg/kg bodyweight of crude methanol extract of
E.camaldulensis (leaf) survived the study period with
29 drug cleared parasites from circulation and survived longer (Fig. 3). There was no significant difference between the activities of the crude extract and the standard drug, but both were significantly different from the activities exhibited by the two fractions
(p≤0.05).

Br. J. Pharmacol. Toxicol., 4(2): 25-32, 2013
Phytochemical compositions of crude methanol extract of E. camaldulensis (leaf) and fractions 8 and
9: Phytochemical analysis of the crude methanol extract of E. camaldulensis (leaf) revealed the presence of terpenes, steroids, saponins, tannins, alkaloids and fatty acids while fraction 8 contained terpenes, steroids and fatty acids and fraction 9 contained terpenes, steroids, saponins, flavonoids, tannins and fatty acids. Alkaloids safety and efficiency much superior to that of its isolated and pure active components (Shariff, 2001).
The importance of these observations and inferences are that: first, the crude extract can be standardised and packaged as phytomedicine, just the way it is done in traditional medical practice. Secondly, the partially purified fractions or their components, though not as were absent in both fractions.
Gas Chromatography-Mass Spectroscopy (GC-MS) analysis: Based on the spectroscopic data from GCactive as the crude extract could be subjected to chemical modification to potentiate their antitrypanosomal activities.
Sub inoculation experiments using blood and CSF
MS, the relative intensities, selectivity index and the peak values of components and deductions made from the National Institute of Science and Technology
(NIST) library, GC-MS analysis indicated components from cured mice did not result in infection thus indicating that the extract succeeded in clearing all blood stream parasites and there was ultimately no infiltration of parasites into the Central Nervous System that resembled 2-Chloro-N-(1, 3-thiozol-2-yl) acetamide, 9-Octadecenamide, 1-Nonadecene, (Z)-9-
Eicosene, Hexadecanol, 1-pentadecanol for fraction 8; and Methylhexadecanoate, Methyl cis-9-octadecenoate,
(CNS). Therefore, this extract, based on the result obtained in this study, is a possible ethno medical candidate for the treatment of early stage trypanosome infection in animals. As for late stage infection, we
Methyl-n-octadecanoate, 1-heptadecanol for fraction 9.
The crude methanol extract of the experimental
DISCUSSION
Trypanosoma brucei brucei three weeks completely cleared parasites from circulation and all animals in the different dose groups survived thereafter for more than three months until they were used for further studies.
E. camaldulensis
(leaf) exhibited very significant activity against infection in mice. The extract when administered consecutively for cannot completely rule out the ability of the crude extract to cross the blood brain barrier (bbb) into the
CNS because when it was administered 72 h post infection, a significant level of parasite clearance was observed (p≤0.05).
An overview of the identified components in fraction 8 shows that 1- Nonadecene and (Z)-9-
Eicosene are sex pheromones in some insects, where they serve as attractants for males (El-Sayed, 2009); hexadecanol, is a commercial product used as a cosmetic ingredient, while excessive exposure to 1pentadecanol produces some central nervous system
The crude methanol extract of E. camaldulensis
(leaf) cleared parasites completely from circulation only when it was administered 24 h after infection, but did not clear parasites completely when treatment was depression and prolonged contact produces skin irritation. It is not clear what role they could be playing in the anti-trypanosomal activity of the crude methanol extract of E. camaldulensis . The case of the other two delayed beyond 24 h post-infection although some antitrypanosomal activity was still observed.
The partially purified fractions (fractions 8 and 9) that exhibited minimal antitrypanosomal activities did components, 2-Chloro-N-(1, 3-thiozol-2-yl) acetamide and 9-Octadecenamide is different because evidence from literature shows that some acetanamides are potent inhibitors of Histone Deacetylase (HDAC), an not completely clear parasites from circulation and the animals died in less than 2 weeks. It was clear from these results that the crude extract as used was more efficacious than the single fractions. We can deduce enzyme responsible for the deacetylation of N-acetyl lysine residues of histone and non-histone proteins
(James and Henry, 2006). And there is growing evidence that the acetylation state of proteins and thus three possible inferences from these observations which are that, the crude extract is trypanocidal, but only at the initial phase of T. b. brucei infection; secondly, fractionation of the crude extract must have resulted in the HDAC enzyme plays a crucial role in the modulation of a number of biological processes, including transcription and cell cycle, which is the basis for their use in the treatment of cancer (Marks et al ., a partial loss of activity and thirdly, there is the possibility of synergistic action between the bioactive components of the crude extract since the fractions, when administered individually, were not as active as
2000). On the basis of this, we may infer that the amides in fraction 8 could be playing the role of inhibitors of certain enzyme (s) unknown to us, but because their concentration in the fraction was small, the crude extract. It has been observed before now that the active components of herbal remedies have the advantage of being combined with many other substances that appear to be inactive. However, these complementary components give the plant as a whole a they elicited a minimal effect on the trypanosomes.
Another aspect of the present study that could be exploited is the treatment of trypanosomiasis by administration of methylating agents. Some methylating agents have been found to be effective against
T. rhodesiense and T. gambiense , which cause fatal
30

diseases in man and also against T. brucei , T. evansi and T. equiperdum , which are of veterinary importance.
That study concerned a method of treating trypanosomiasis in patients, e.g., warm-blooded animals, such as humans, horses, sheep, goats, swine, camels or cattle, by administering to such patients a trypanocidal effective amount of a methylating agent of the formula CH group, e.g., OH or SO more particularly an unsubstituted or substituted alkyl having 1 to 10, preferably 1 to 6, carbon atoms or an unsubstituted or substituted aryl, including other species capable of generating methyl radicals (CH diazomethane (CH
N
2
+
) (Shyam demonstrated that repeated exposure of trypanosomes in vitro or
3
N = NX', wherein X' is a leaving
2
N et al in vivo
2
R'" and R'" an alkyl or an aryl,
2
3
●
) or methyldiazonium (CH
),
3
., 1987). It has also been
to low concentrations of the methylating agent 1,2-bis methylhydrazine induces a series of moderately synchronous morphological and biochemical changes.
Cell division halts and the long-slender bloodstream forms transform to short-stumpy forms via larger intermediate-stage cells which contain approximately double the normal G
2
content of DNA (Philip et al .,
1991).
Methyl hexadecanoate, Methyl-cis-9-octadecanoate and
Methyl-n-octadecanoate, which might be likely methylating agents that could play positive roles in the therapy of African trypanosomiasis. Literature indicates that Methyl-cis-9-octadecenoate was first identified in the cerebrospinal fluid of sleep-deprived cats, rats and humans. It induced physiological sleep when injected into rats intraperitoneally at 5-50 mg doses (Cravatt et al
Br. J. Pharmacol. Toxicol., 4(2): 25-32, 2013
(methylsulfonyl)-1-
Interestingly, fraction 9 contains compounds like
., 1995). Methylating agents appear to have two major effects on trypanosomes, depending upon the dose level. At high levels, cytokinesis appears to be inhibited almost immediately and the cells are transformed into transitional forms containing multiple nuclei and kinetoplasts. These cells disappear from the bloodstream in 48 to 72 h. When administered at repetitive low doses, methylating agents induce the entire population to differentiate into short-stumpy forms (short-stumpy forms cannot differentiate further unless they are taken up by a feeding tsetse fly or placed in appropriate culture conditions), as judged by morphology, NADH diaphorase positivity and other biochemical and physiological criteria. Short-stumpy forms are non-dividing differentiated cells and are not infective to the mammalian host. The latter property may make these agents useful biochemical tools in the study of differentiation in trypanosomes, since, with these compounds, it is possible to induce the entire population of trypanosomes to differentiate in a moderately synchronous manner and through this approach early events in the differentiation process can be studied. Both single high dose regiments and repetitive low doses can result in cures of trypanosomiasis using a number of the methylating agents (Philip et al ., 1991).
Difluoro Methyl Ornithine (DFMO) or eflornithine has also been shown to induce differentiation in
T. brucei (Goldberg et al ., 1997). This effect is generally attributed to the depletion of polyamines. The depletion of polyamines and trypanothione as a result of the DFMO treatment may potentiate the actions of
SAM and DSAM as methylating agents by decreasing the levels of competing nucleophiles. Depletion of polyamines may also make the nucleic acids more susceptible to methylation (Fairlamb et al ., 1983). SAM is also the methyl donor used by many methylases; therefore, enzymatically - mediated - methylation reactions may also be affected. Although methylating agents in general are mutagenic, in cases of multi-drug resistant trypanosomiasis which have failed to respond to existing therapies, these compounds may be extremely effective. The distinct advantages of methylating agents over existing trypanocides include:
•
High therapeutic indices
•
Oral activity
•
Novel mechanism of action
•
Broad spectrum antitrypanosomal activity
•
Favourable pharmacokinetics which makes these compounds candidates for both agricultural and clinical development (Fairlamb et al ., 1983).
From the foregoing, it can be deduced that the minimal antitrypanosomal activity exhibited by fraction
9 may be due to the presence of three potential methyl donors and the relatively low concentration of these agents prevented complete clearance of parasites from the circulation of infected mice.
The anti-trypanosomal potential of the methanol extract of E. camaldulensis has been demonstrated in this study and may give credence to the acclaimed therapeutic potency of E. camaldulensis (leaf) as an anaesthetic, antiseptic, astringent and as a folk remedy for colds, colic, coughs, diarrhoea, dysentery, haemorrhage, laryngalgia, laryngitis, pharyngitis, sore throat, spasm, trachalgia and wounds (Batish et al .,
2008) and its use locally in combination with other plants as a remedy for fever in some Nigerian communities.
It could be concluded from the result obtained in this study that the crude methanol extract of
E. camaldulensis (leaf) is a potential candidate for the treatment of African Animal Trypanosomiasis because it is effective, cheap and is not acutely toxic and the plant is readily available.
ACKNOWLEDGMENT
This study was funded by a research grant to
Emmanuel O. Ogbadoyi by the Organization for the
Prohibition of Chemical Weapons (Hague) and a
Science and Technology Education-Post Basic (STEP-
31

Br. J. Pharmacol. Toxicol., 4(2): 25-32, 2013
B) grant to the Federal University of Technology,
Minna, Nigeria.
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